Poliovirus 2A<sup>pro</sup> expression inhibits growth of yeast cells

Barco, Ángel; Carrasco, Luís

doi:10.1016/0014-5793(95)00841-v

Cited by 16 publications

(15 citation statements)

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“…Expression of 2APm in yeast cells resulted in a marked decrease in protein synthesis and a change in morphology (Barco & Carrasco, 1995;Klump et al, 1996). However, the yeast homologue of mammalian eIF-4G was found to be intact, indicating that the effects on yeast cells have to be attributed to cleavage of so-far unknown proteins.…”

Section: G859mentioning

confidence: 99%

Cleavage site analysis in picornaviral polyproteins: Discovering cellular targets by neural networks

et al. 1996

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Picornaviral proteinases are responsible for maturation cleavages of the viral polyprotein, but also catalyze the degradation of cellular targets. Using graphical visualization techniques and neural network algorithms, we have investigated the sequence specificity of the two proteinases 2AP" and 3CPm. The cleavage of VPO (giving rise to VP2 and VP4). which is carried out by a so-far unknown proteinase, was also examined. In combination with a novel surface exposure prediction algorithm, our neural network approach successfully distinguishes known cleavage sites from noncleavage sites and yields a more consistent definition of features common to these sites. The method is able to predict experimentally determined cleavage sites in cellular proteins. We present a list of mammalian and other proteins that are predicted to be possible targets for the viral proteinases. Whether these proteins are indeed cleaved awaits experimental verification. Additionally, we report several errors detected in the protein databases.A computer server for prediction of cleavage sites by picornaviral proteinases is publicly available at the e-mail address NetPicoRNA@cbs.dtu.dk or via WWW at http://www.cbs.dtu.dkfservices/NetPicoRNA.Keywords: cleavage site prediction; neural networks; picornavirus; proteinase; surface exposureMembers of the picornavirus family express their genomic RNA as a single polyprotein that is proteolytically processed to the mature polypeptides. At least three proteinases are required for the individual protein components to be released (reviewed in Krausslich Hellen et al., 1989;Lawson & Semler, 1990). The primary cleavage, which severs the capsid precursor PI from the nonstructural region P2-P3, is performed cotranslationally by the viral proteinase 2APm in enteroviruses and human rhinoviruses (HRVs; see Fig. I). Most of the remaining cleavages are catalyzed by the viral proteinase, 3CP". In cardio-, hepato-, and aphthoviruses, which also belong to the picornavirus family, the L-proteinase performs functions similar to those of 2APm, resulting in a somewhat different cleavage scheme (see Fig. I). Concomitantly with RNA encapsidation, VPO is cleaved to VP4 and VP2; it is believed that the RNA itself exerts a catalytic function in this event (Arnold et a!., 1987;Harber et al., 1991;Bishop &Anderson, 1993;Basavappa et al., 1994).In addition to processing of the viral polyprotein, the proteinases also cleave cellular targets. When infected with poliovirus, at least nine acidic and five basic cellular proteins were shown to be degraded in two-dimensional gel electrophoresis (Urzanqui & Carrasco, 1989). The degradation of cellular proteins seems to be part of the viral attack mechanism, leading to host cell shur-ofJ-a decrease in cellular transcription and translation that has no influence on viral replication. The best-studied event is the cleavage of the eukaryotic initiation factor 4G (eIF-4G). which is required for cap-dependent translation of cellular mRNA. This protein is degraded by 2APm in entero-and...

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Section: G859mentioning

confidence: 99%

Cleavage site analysis in picornaviral polyproteins: Discovering cellular targets by neural networks

et al. 1996

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“…The potential cellular toxicity of the individual expression of 2A pro has been analyzed in different cellular systems, including bacteria where this protease was nontoxic (4,48), yeast (7,8,31), and mammalian cells. A variety of expression systems have been examined in cultured cells.…”

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A Stable HeLa Cell Line That Inducibly Expresses Poliovirus 2A ^pro : Effects on Cellular and Viral Gene Expression

Barco

Feduchi

Carrasco

2000

J Virol

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“…Inducible expression of poliovirus nonstructural proteins in the yeast Saccharomyces cerevisiae showed that two of them, namely 2A pro and 2BC, inhibited yeast growth (32)(33)(34). Recently, a similar effect was found for rhinovirus 2A pro (35).…”

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confidence: 79%

“…Hydroxylamine Mutagenesis and Genetic Assay-The plasmid pEMBL.2A (32) was randomly mutated using hydroxylamine as described (36). This mutated DNA was used to transform the S. cerevisiae strain W303-1B (Mat ␣, Ade2, His3, Leu2, Trp1, Ura3) to obtain 100 -200 colonies in each YNB.Glu plate.…”

Section: Methodsmentioning

confidence: 99%

“…This would provide additional data on the interactions of different regions of the 2A pro . Although poliovirus 2A pro blocks S. cerevisiae growth strongly, the exact target of its activity remains unknown (32). Although gene expression in yeast cells is clearly affected, the target recognized by 2A pro is probably unrelated to mammalian eIF-4G (35 pro is a translational activator of mRNAs bearing the poliovirus IRES sequence (22) and is involved in viral RNA replication (26,27).…”

Section: S Cerevisiae As a System For Obtaining Poliovirus 2amentioning

confidence: 99%

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The Yeast Saccharomyces cerevisiae as a Genetic System for Obtaining Variants of Poliovirus Protease 2A

Barco¹,

Ventoso²,

Carrasco

1997

Journal of Biological Chemistry

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pro sequences from these colonies contain point mutations in the poliovirus protease. The different variant protease sequences were transferred to an infectious poliovirus cDNA clone. Translation of genomic RNA obtained from these poliovirus mutants in cell-free systems revealed that some of them had defects in their ability to cleave P1-2A in cis. In addition, several of these variants cleaved the translation initiation factor eIF-4G inefficiently. Transfection of the RNA generated from the full-length poliovirus genomes mutated in 2A pro yielded five viable polioviruses with a small plaque phenotype. These five polioviruses efficiently cleaved p220 but showed defects in viral protein synthesis, transactivation of a leader-luciferase mRNA, and 3CD cleavage to 3C and 3D. All 2A pro mutant sequences, including those that did not yield viable viruses, were cloned in pTM1 vector under a T7 promoter. Only the 2A pro variants that have activity to cleave 3CD produced viable poliovirus. Our findings indicate that S. cerevisiae represents a useful system for obtaining poliovirus 2A pro variants that may provide further insight into the role of this protease during the poliovirus replication cycle.

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Poliovirus 2A^pro expression inhibits growth of yeast cells

Cited by 16 publications

References 34 publications

Cleavage site analysis in picornaviral polyproteins: Discovering cellular targets by neural networks

Cleavage site analysis in picornaviral polyproteins: Discovering cellular targets by neural networks

A Stable HeLa Cell Line That Inducibly Expresses Poliovirus 2A ^pro : Effects on Cellular and Viral Gene Expression

The Yeast Saccharomyces cerevisiae as a Genetic System for Obtaining Variants of Poliovirus Protease 2A

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Poliovirus 2Apro expression inhibits growth of yeast cells

Cited by 16 publications

References 34 publications

Cleavage site analysis in picornaviral polyproteins: Discovering cellular targets by neural networks

Cleavage site analysis in picornaviral polyproteins: Discovering cellular targets by neural networks

A Stable HeLa Cell Line That Inducibly Expresses Poliovirus 2A pro : Effects on Cellular and Viral Gene Expression

The Yeast Saccharomyces cerevisiae as a Genetic System for Obtaining Variants of Poliovirus Protease 2A

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Poliovirus 2A^pro expression inhibits growth of yeast cells

A Stable HeLa Cell Line That Inducibly Expresses Poliovirus 2A ^pro : Effects on Cellular and Viral Gene Expression